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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-05-1307.
Submitted May 2, 2002
Accepted June 26, 2002
Identification of Distal Regulatory Regions in the Human  IIb Gene Locus Necessary for Consistent, High-Level Megakaryocyte Expression
Michael A Thornton, Chunyan Zhang, Maria A Kowalska, and Mortimer Poncz*
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Pediatrics, University of Pennsylvania, Philadelphia, PA, USA
Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA, USA
* Corresponding author; email: poncz{at}email.chop.edu.
The IIb/ß3 integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of IIb, and studies of the IIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the IIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) IIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the IIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive sites (HS) that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the hIIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue-specificity, but that a region 2.5 kb to 7.1 kb upstream of the hIIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold, and lead to levels of hIIb mRNA that were ~30% of the native mIIb mRNA level. These constructs also resulted in detectable hIIb/mß3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the IIb gene for tissue-specificity, but also characterizes the distal organization of the IIb gene locus and provides an initial localization of two important regulatory regions needed for the expression of the IIb gene at high levels during megakaryopoiesis.
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